Biological systems for manufacture of polyhydroxyalkanoate polymers containing 4-hydroxyacids

ABSTRACT

The gene encoding a 4-hydroxybutyryl-CoA transferase has been isolated from bacteria and integrated into the genome of bacteria also expressing a polyhydroxyalkanoate synthase, to yield an improved production process for 4HB-containing polyhydroxyalkanoates using transgenic organisms, including both bacteria and plants. The new pathways provide means for producing 4HB containing PHAs from cheap carbon sources such as sugars and fatty acids, in high yields, which are stable. Useful strains are obtaining by screening strains having integrated into their genomes a gene encoding a 4HB-CoA transferase and/or PHA synthase, for polymer production. Processes for polymer production use recombinant systems that can utilize cheap substrates. Systems are provided which can utilize amino acid degradation pathways, α-ketoglutarate, or succinate as substrate.

This application is a continuation of U.S. Ser. No. 10/006,915 filedNov. 9. 2001 now U.S. Pat No. 6,689,589, which is a continuation of U.S.Ser. No. 09/156,809 filed Sep. 18, 1998 (now U.S. Pat. No. 6,316,262),which claims priority to U.S. Ser. No. 60/059,373 filed Sep. 19, 1997,entitled Biological Systems for the Manufacture of PolyhydroxyalkanoatePolymers containing 4-Hydroxyacids by Gjalt W. Huisman, Frank A. Skraly,David P. Martin, and Oliver P. Peoples.

BACKGROUND OF THE INVENTION

Poly [(R)-3-hydroxyalkanoates] (PHAs) are biodegradable andbiocompatible thermoplastic materials, produced from renewableresources, with a broad range of industrial and biomedical applications(Williams and Peoples, 1996, CHEMTECH 26, 38–44). In recent years, whatwas viewed as a single polymer, poly-β-hydroxybutyrate (PHB), hasevolved into a broad class of polyesters with different monomercompositions and a wide range of physical properties. To date around onehundred different monomers have been incorporated into the PHA polymers(Steinbüchel and Valentin, 1995, FEMS Microbiol. Lett. 128; 219–228). Ithas been useful to broadly divide the PHAs into two groups according tothe length of their side chains and their pathways for biosynthesis.Those with short side chains, such as polyhydroxybutyrate (PHB), ahomopolymer of R-3-hydroxybutyric acid units,—OCR¹R²(CR³R⁴)_(n)CO—

-   -   where: n is 0 or an integer and R¹, R², R³, and R⁴ are each        selected from saturated and unsaturated hydrocarbon radicals;        hal- and hydroxy-substituted radicals; hydroxy radicals; halogen        radicals; nitrogen-substituted radicals; oxygen-substituted        radicals; and hydrogen atoms,        are crystalline thermoplastics, whereas PHAs with long side        chains are more elastomeric. The former have been known for        about seventy years (Lemoigne & Roukhelman, 1925), whereas the        latter materials were first identified in the early 1980's        (deSmet et al., 1983, J. Bacteriol., 154; 870–878). Before this        designation, however, PHAs of microbial origin containing both        (R)-3-hydroxybutyric acid and one or more long side chain        hydroxyacid units containing from five to sixteen carbon atoms        had been identified (Steinbüchel and Wiese, 1992, Appl.        Microbiol. Biotechnol. 37: 691–697; Valentin et al., 1992, Appl.        Microbiol. Biotechnol. 36: 507–514; Valentin et al., 1994, Appl.        Microbiol. Biotechnol. 40: 710–716; Lee et al., 1995, Appl.        Microbiol. Biotechnol. 42: 901–909; Kato et al., 1996, Appl.        Microbiol. Biotechnol. 45: 363–370; Abe et al., 1994, Int. J.        Biol. Macromol. 16: 115–119; Valentin et al., 1996, Appl.        Microbiol. Biotechnol. 46: 261–267; U.S. Pat. No. 4,876,331). A        combination of the two biosynthetic pathways probably provide        the hydroxyacid monomers. These latter copolymers can be        referred to as PHB-co-HX. Useful examples of specific        two-component copolymers include PHB-co-3-hydroxyhexanoate        (Brandl et al., 1989, Int. J. Biol. Macromol. 11; 49–55; Amos        and McInerey, 1991, Arch. Microbiol. 155: 103–106; Shiotani et        al., 1994, U.S. Pat. No. 5,292,860). Chemical synthetic methods        have also been used to prepare racemic PHB copolymers of this        type for applications testing (WO 95/20614, WO 95/20615 and WO        96/20621).

Numerous microorganisms have the ability to accumulate intracellularreserves of PHA polymers. Since polyhydroxyalkanoates are naturalthermoplastic polyesters, the majority of their applications are asreplacements for petrochemical polymers currently in use for packagingand coating applications. The extensive range of physical properties ofthe PHA family of polymers, in addition to the broadening of performanceobtainable by compounding and blending as traditionally performed in thepolymer industry, provides a corresponding broad range of potentialend-use applications. The PHAs can be produced in a wide variety oftypes depending on the hydroxyacid monomer composition (Steinbüchel andValentin, 1995, FEMS Microbiol. Lett. 128: 219–228). This wide range ofpolymer compositions reflects an equally wide range of polymer physicalproperties including: a range of melting temperatures from 40° C.–180°C., glass transition temperatures from −35 to 5° C., degrees ofcrystallinity of 0% to 80% coupled with the ability to control the rateof crystallization and elongation to break of 5 to 500%.Poly(3-hydroxybutyrate), for example, has characteristics similar tothose of polypropylene while poly(3-hydroxyoctanoate) (a copolymer of(R)-3-hydroxyoctanoate and (R)-3-hydroxyhexanoate) types behave more aselastomers and PHAs with longer side chains giving behavior closer towaxes. The PHAs can also be plasticized and blended with other polymersor agents. One particularly useful form is as a latex of PHA in water.

The monomer compositions also affect solubility in organic solventsallowing for a choice of a wide range of solvents. Copolymers of(R)-3-hydroxybutyrate and other hydroxyacid comonomers havesignificantly different solubility characteristics from those of the PHBhomopolymer.

To date, PHAs have seen limited commercial availability with only thecopolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) beingavailable in significant quantities. This copolymer has been produced byfermentation of the bacterium Ralstonia eutropha (formerly Alcaligeneseutrophus). Fermentation processes for other PHAs have been developed(Williams and Peoples, 1996, CHEMTECH 26: 38–44). Plant crops are alsobeing genetically engineered to produce these polymers, and offer a coststructure in line with the vegetable oils and direct pricecompetitiveness with petroleum based polymers (Williams and Peoples1996, CHEMTECH 26: 38–44). More traditional polymer synthesis approacheshave also been examined, including direct condensation and ring-openingpolymerization of the corresponding lactones (Jesudason andMarchessault, 1994, Macromolecules 27: 2595–2602, U.S. Pat. No.5,286,842; U.S. Pat. No. 5,563,239; U.S. Pat. No. 5,516,883; U.S. Pat.No. 5,461,139; Canadian patent application 2,006,508).

Synthesis of PHA polymers containing the monomer 4-hydroxybutyrate(PHB4HB, Doi, Y. 1995, Macromol. Symp. 98, 585–599) or 4-hydroxyvalerateand 4-hydroxyhexanoate containing PHA polyesters have been described(Valentin et al., 1992, Appl. Microbiol. Biotechnol. 36: 507–514 andValentin et al., 1994, Appl. Microbiol. Biotechnol. 40: 710–716). Thesepolyesters have been manufactured using methods similar to thatoriginally described for PHBV in which the microorganisms are fed arelatively expensive non-carbohydrate feedstock in order to force theincorporation of the monomer into the PHA polyester. For example,production of PHB4HB has been accomplished by feeding glucose and4-hydroxybutyrate or substrate that is converted to 4-hydroxybutyrate toA. eutrophus (Kunioka, M., Nakamura, Y., and Doi, Y. 1988, Polym.Commun. 29: 174; Doi, Y., Segawa, A. and Kunioka, M. 1990, Int. J. Biol.Macromo. 12: 106; Nakamura, S., Doi, Y. and Scandola, M. 1992,Macromolecules 25: 423), A. latus (Hiramitsu, M., Koyama, N. and Doi, Y.1993, Biotechnol. Lett. 15: 461), Pseudomonas acidovorans (Kimura, H.,Yoshida, Y. and Doi, Y. 1992, Biotechnol. Lett. 14: 445) and Comomonasacidovorans (Saito, Y. and Doi, Y., 1994, Int. J. Biol. Macromol. 16:18). Substrates that are converted to 4-hydroxybutyrate are1,4-butanediol, 1,6-hexanediol, 1,8-octanediol, 1,10-decanediol,1,12-dodecanediol and 1,4-butyrolactone. The PHB4HB copolymers can beproduced with a range of monomer compositions which again provides arange of polymer properties. In particular as the amount of 4HBincreases above 10 wt. %, the melting temperature (T_(m)) decreasesbelow 130° C. and the elongation to break increases above 400% (Saito,Y., Nakamura, S., Hiramitsu, M. and Doi, Y., 1996, Polym. Int. 39: 169).

The formation of 4HB containing polymers has also been studied withrecombinant strains in studies aimed at improved PHB-4HB formation inRalstonia eutropha or E. coli. Mutants of R. eutropha H16 were selectedthat cannot use 4-hydroxybutyrate as a carbon source. When such mutantswere tested for copolymer formation, up to 84% 4HB was incorporated intothe accumulated PHA (Kitamura S and Y. Doi, 1994. in BiodegradablePlastics and Polyesters, 12, p. 373–378). By introducing additionalcopies of the phb genes, the accumulation of PHB-4HB was enhanced (Lee,Y.-H., Park, J.-S. and Huh, T.-L. 1997, Biotechnol. Lett. 19: 771–774).

It is desirable to develop more cost effective ways of producing PHAscontaining 4HB by biological systems. Several factors are critical foreconomic production of PHA: substrate costs, fermentation time, andefficiency of downstream-processing. A general characteristic of theabove described bacteria is that their growth rate is low, they areoften difficult to break open and their amenity to genetic engineeringis limited. Therefore, processes have been developed that improve theeconomics of PHA production by using transgenic organisms. Formation ofPHB4HB was achieved in E. coli using the 4-hydroxybutyrate pathway fromC. kluyveri (Hein, S., Söhling, B., Gottschalk, G., and Steinbüchel, A.1997. FEMS Microbiol. Lett. 153: 411–418). In these studies both the4-hydroxybutyryl-CoA transferase and PHA synthase were plasmid encoded.Subsequent work showed that the 4-hydroxybutyrate pathway from C.kluyveri supports formation of PHB-4HB in E. coli up to 50% of the celldry weight from glucose as sole carbon source, and where 2.8% of themonomers is 4HB. The 4HB monomer in these strains is most likely derivedfrom succinate, an intermediate of the TCA cycle (Valentin, H. E. andDennis, D. 1997. J. Biotechnol. 58: 33–38). These studies were based onEscherichia coli as recombinant production organisms and PHAbiosynthetic genes from PHA producers such as R eutropha.

It is an object of the present invention to provide recombinantprocesses whereby additional genes can be introduced in transgenic PHBproducers to create new strains that synthesize monomers, such as 4HB,for alternative PHAs.

A further object of the present invention is to provide techniques andprocedures to stably engineer transgenic organisms that synthesize PHAscontaining 4-hydroxybutyrate either as sole constituent or asco-monomer.

It is also an object of the present invention to provide screeningsystems for new 4-hydroxybutyryl CoA transferase encoding genes.

It is another object of the present invention to provide techniques andprocedures to engineer new pathways in biological systems for theendogenous synthesis of alternative PHA monomers.

SUMMARY OF THE INVENTION

Improved production processes for 4HB containing PHAs using transgenicstrains have been developed. Transgenic E. coli strains are described inwhich the required phb genes have been integrated on the chromosome.Additional genes for the synthesis of the 4HB monomer are alsointegrated on the chromosome. The latter genes can be derived from abroad range of organisms which carry a 4-hydroxybutyryl-CoA transferaseand be identified by screening for this activity in the engineered E.coli strains described here. In addition, an endogenous E. coli activityis disclosed that can be further improved for the purpose of 4HB-CoAtransferase activity. New pathways are also disclosed for the supply ofintermediates of 4HB biosynthetic pathways such as α-ketoglutarate andγ-aminobutyrate. The diversity of these pathways is important for thesuccessful production of 4HB containing PHAs from cheap carbon sourcessuch as sugars and fatty acids.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is the alignment of the C. kluyveri OrfZ sequence with theN-terminal sequence and internal sequences of 4-hydroxybutyryl CoAtransferase (4HBCT) from C. aminobutyricum (SEQ ID Nos 1 and 2.Identical residues are indicated, similar residues are indicated by *.FIGS. 1B and 1C are the nucleotide sequence of the orfZ gene from C.kluyeri. FIG. 1D is the amino acid sequence of the orfZ gene from C.kluyeri.

FIG. 2 is a schematic of the endogenous synthesis of 4-hydroxybutyrylCoA from α-ketoglutarate through the GABA shunt. 1. α-ketoglutarateaminotransferase; 2. glutamate decarboxylase; 3. GABA transaminase; 4.Succinic semialdehyde reductase; 5. 4-hydroxybutyryl CoA transferase.

FIG. 3 is a schematic of the endogenous synthesis of4-hydroxybutyryl-CoA from GABA precursors. GABA is an intermediate inthe degradation of amino acids such as arginine, glutamine and proline.Genes in arginine degradation are encoded by speA, adi, speB, pat andprr; genes in glutamine degradation are encoded by gltBD and gadB, genesin proline degradation are encoded by putA and gadB. GABA is convertedto 4-hydroxybutyryl-CoA by the gene products of gabT, 4hbD and hbcT.

FIG. 4 is a schematic of the endogenous synthesis of 4-hydroxybutyrylCoA from succinate. 1. succinyl CoA-CoA transferase; 2. succinatesemialdehyde dehydrogenase; 3. 4-hydroxybutyrate dehydrogenase; 4.4-hydroxybutyryl CoA transferase.

FIG. 5 is a schematic of the construction of plasmids for integration ofthe PHB synthase (phbC) gene from Z. ramigera into the chromosome of E.coli and other Gram-negative bacteria.

FIG. 6 and FIG. 6A are a schematic of the construction of plasmids forintegration of 3-ketoacyl-CoA thiolase (phbA) and acetoacetyl-CoAreductase (phbB) genes from Z. ramigera into the chromosome of E. coliand other Gram-negative bacteria.

FIG. 7 is a schematic of the metabolic and genetic representation of theengineered biosynthetic pathway for 4-hydroxybutyryl-CoA synthesis. Thegene products of gabT, 4hbD and hbcT are required for this pathway,gadAB and gdhA are helpful, whereas the gene products of aspC, sad andgabD are preferably absent or inactive.

FIG. 8 is a schematic of the construction of plasmids pMSX-TD andpMSXTp1-TD, which expresses enzymes to convert α-ketoglutarate to4-hydroxybutyryl-CoA.

FIG. 9 is a schematic of the construction of plasmids pMSX-ABT,pMSXTp1-ABT and pMSXTp1-BT, which expresses enzymes to convertα-ketoglutarate to 4-hydroxybutyryl-CoA.

FIG. 10 is a schematic of the construction of plasmid pMSX-ABT andpMSX-ABT-TD which expresses enzymes to convert α-ketoglutarate to4-hydroxybutyryl-CoA.

FIG. 11 is a schematic of the construction of plasmid pMSX-T1DD whichexpresses enzymes to convert succinate to 4-hydroxybutyryl-CoA

DETAILED DESCRIPTION OF THE INVENTION

The minimal biological requirement for the synthesis ofpoly(3-hydroxybutyrate-co-4-hydroxybutyrate) have been defined.Enzymatic synthesis of the substrates for PHA synthase from R. eutrophawas achieved by incubation of equimolar amounts of (R)-3-hydroxybutyrateand 4-hydroxybutyrate with 4-hydroxybutyrate CoA transferase. In situmonomer-CoA synthesis coupled by direct enzymatic polymerization resultsin the formation of a PHB-4HB copolymer as determined by ¹H-NMR of theresulting polymer. Techniques and procedures to engineer transgenicorganisms that synthesize PHAs containing 4-hydroxybutyrate either assole constituent or as co-monomer have been developed. In these systemsthe transgenic organism is either a bacterium eg. Escherichia coli, K.pneumoniae, Ralstonia eutropha (formerly Alcaligenes eutrophus),Alcaligenes latus or other microorganisms able to synthesize PHAs, or ahigher plant or plant component, such as the seed of an oil crop(Brassica, sunflower, soybean, corn, safflower, flax, palm or coconut orstarch accumulating plants (potato, tapioca, cassava). A screeningprocedure for the identification of genes encoding enzymes capable ofconverting 4-hydroxybutyric acid to 4-hydroxybutyryl-CoA and methods forredirecting the flux of normal cellular metabolites such as e.g.succinic acid and/or glutamic acid to 4-hydroxybutyric acid has beendeveloped. The gene encoding a 4-hydroxybutyryl CoA transferase genefrom the Gram-positive, strict anaerobic bacterium Clostridium kluyverihas been identified and used to express this enzyme activity in atransgenic organism to convert 4-hydroxybutyric acid into4-hydroxybutyryl-CoA resulting in the accumulation ofpoly(4-hydroxybutyrate) in E. coli. A bacteria expressing a functionalPHA synthase from a transgene is described, as well as methods forexpressing these genes in transgenic plant crops.

Screening systems for new 4-hydroxybutyryl CoA transferase encodinggenes are also described. Transgenic E. coli strains in which a PHAsynthase encoding gene is integrated in the chromosome and expressed tolevels supporting PHA synthesis have been developed. With thesetransgenic strains can be screened with genomic libraries from differentbiological sources for activities that convert alternative PHAprecursors such as 4-hydroxybutyrate to corresponding substrates for PHAsynthase.

Techniques and procedures are provided to engineer new pathways inbiological systems for the endogenous synthesis of alternative PHAmonomers. Metabolism of any PHA production organism, including bacteriaand plant crops, can be redirected to supply specific metabolites forPHA synthesis by metabolic engineering. In order to make this approacheffective, it is necessary to develop new biochemical pathways leadingto the desired monomer from one of the common metabolic intermediates.It is not necessary that such pathways exist in one organism since theindividual steps can be reconstituted in the production organism ofchoice using genetic engineering techniques.

Incorporation of alternative monomers derived from supplementedfeedstocks has specific drawbacks. First, additional feeds into afermenter are costly as they expand the infrastructure and imposeadditional quality control. Second, addition of monomer precursors needsto be tightly controlled to achieve a constant composition of themonomer pools and PHA composition. Methods to engineer E. coli such atP(4HB) or PHB-co-4HB synthesis occurs from inexpensive carbohydratefeedstocks such as glucose, sucrose, xylose and lactose as the onlycarbon source. Enzyme activities in the γ-hydroxybutyrate shunt areelevated, while enzyme activities that drain intermediates from thisshunt are reduced. An alternative pathway yields 4HB from succinate. Asimilar approach in metabolic engineering can accommodate production of4HB containing PHAs in organisms such as A. eutrophus, A. latus andComamonas which are currently capable of producing 4-hydroxybutyratecopolymers from cosubstrates and in transgenic microbial and plant cropsystems expressing a PHA synthesis from a heterologous PHA synthase geneor genes.

It is crucial for efficient PHA synthesis in recombinant E. coli strainsthat the expression of all the genes involved in the pathway beadequate. To this end, the genes of interest can be expressed fromextrachromosomal DNA molecules such as plasmids, which intrinsicallyresults in a copy number effect and consequently high expression levels,or, more preferably, they can be expressed from the chromosome. Forlarge scale fermentations of commodity type products it is generallyknown that plasmid-based systems are unsatisfactory due to the extraburden of maintaining the plasmids and the problems of stableexpression. These drawbacks can be overcome using chromosomally encodedenzymes by improving the transcriptional and translational signalspreceding the gene of interest such that expression is sufficient andstable.

Production of 4HB Copolymers

Gerngross and Martin reported that substrates of PHA synthase requirethe presence of a coenzyme A (CoA) moiety (Gerngross, T. U. and Martin,D. P. (1955) Proc. Natl. Acad. Sci. USA 92:6279). The precursor requiredfor the incorporation of 4HB is therefore 4HB-CoA. To determine theminimal requirement for the synthesis of 4-hydroxybutyrate containingPHAs, a mixture of 4-hydroxybutyrate, 3-hydroxybutyrate,4-hydroxybutyrate CoA transferase purified from Clostridiumacelobutylicum (Willadsen and Buckel, FEMS Microbiol. Lett. (1990) 70:187–192) and PHB synthase (as purified by Gerngross et al. (1994)Biochemistry 33:9311) was incubated in vitro under conditions asdescribed by Gerngross and Martin (Gerngross, T. U. and Martin, D. P.(1995) Proc. Natl. Acad. Sci. USA 92:6279. The product of the reactionwas isolated and the incorporation of 4-hydroxybutyrate was confirmed by1H-NMR.

Having established the minimal requirements for the synthesis of4-hydroxybutyrate containing PHA in vitro, it becomes evident that theminimal requirements for the synthesis of these PHAs in vivo includes agene encoding 4-hydroxybutyrate CoA transferase or similar activity and4-hydroxybutyrate. The substrate 4-hydroxybutyrate can be administeredto the PHA producing microorganism or be synthesized in vivo byengineered biosynthetic pathways from appropriate substrates. Amino acidsequence was determined for the purified 4-hydroxybutyrate CoAtransferase (Scherf and Buckel, Appl. Environ. Microbiol. (1991)57:2699–2701). The purified protein was subjected to enzymatic digestionfollowed by amino acid sequence analysis of three of the resultingpeptides. The amino acid sequence of these peptides and the N-terminusof the intact protein showed a striking homology to the OrfZ geneproduct (FIGS. 1A, 1B, 1C and 1D), whose identity and function was notknown, thereby identifying orfZ as the gene encoding 4-hydroxybutyrylCoA transferase in C. kluyveri. This gene was renamed hbcT.

Confirmation that introduction of this gene into an E. coli strain thatexpresses PHB synthase is sufficient for 4-hydroxybutyrate containingPHA synthesis was obtained as follows. The PHB synthase from Z. ramigerais expressed from a chromosomally integrated copy of this gene in E.coli strain MBX379. PHA was formed within the cells upon introduction ofa plasmid encoding hbcT and supplying 4-hydroxybutyrate in the growthmedium. In the absence of genes providing other enzymes of the PHBpathway, the accumulated PHA is P4HB. E. coli strain MBX777 contains thegenes encoding β-ketothiolase, acetoacetyl CoA reductase and PHBsynthase from Z. ramigera. Upon introduction of a plasmid encoding hbcTand supplying 4-hydroxybutyrate in the growth medium, a PHB-4HBcopolymer was formed.

Further development of a PHB-4HB producing system is achieved byengineering the metabolic pathways of the transgenic organism such that4-hydroxybutyrate is synthesized from endogenous intermediates insteadof being supplied externally. Two biochemical routes to the precursor4HB-CoA can be established in a production organism for 4HB-containingPHAs. The first pathway proceeds from α-ketoglutarate, the second fromsuccinate. Substrate for both pathways can also be provided throughamino acid degradation.

Pathway to 4-hydroxybutyryl CoA from α-ketoglutarate

A pathway that enables the conversion of α-ketoglutarate to4-hydroxybutyryl CoA is shown in FIG. 2. Enzymes involved in thispathway are α-ketoglutarate transaminase, glutamate dehydrogenase,glutamate decarboxylase, 4-hydroxybutyrate dehydrogenase and4-hydroxybutyrate CoA transferase.

Genes encoding these activities can be acquired from multiple sources:

gdhA gene encoding glutamate dehydrogenase: E. coli (Valle et al. Gene(1984) 27: 193–199 and Valle et al., Gene (1983) 23: 199–209),Klebsiella aerogenes (Mountain et al., Mol. Gen. Genet. (1985)199:141–145), Pyrococcus furiosus (DiRuggiero et al., Appl. Environ.Microbiol. (1995) 61: 159–164; Eggen et al., Gene (1993) 132:143–148),Sulfolobus shibatae (Benachenhou et al. (1994), Gene 140: 17–24),Rumonococcus flavefaciens (Duncan et al., Appl, Environ. Microbiol.(1992) 58: 4032–4037), Pseudomonas fluorescens (Miyamoto et al., J.Biochem. (1992) 112:52–56), Clostridium symbiosum (Teller et al., Eur.J. Biochem. (1992) 206: 151–159), Synechocystis (Plant Mol. Biol. (1995)28: 173–188), Corynebacterium glulamicum (Bormann et al., Mol.Microbiol. (1992) 6:301–308), Peptostreptococcus asaccharolyticus(Snedecor et al. (1991) J. Bacteriol. 173: 6162–6167), Salmonellatyphimurium (Miller et al. (1984) J. Bacteriol. 157: 171–178), Chlorellasorokiniana (Cock et al., Plant Mol. Biol. (1991) 17: 1023–144),Saccharomyces cerevisiae (Nagasu et al., Gene (1984) 37:247–253),Neurospora crassa (Kinnaird et al., Gene (1983) 26:253–260), Giardialamblia (Yee et al (1992) J. Biol. Chem. 267: 7539–7544).

gadA and/or gadB encoding glutamate-succinic semialdehyde transaminase:E. coli (Metzer and Halpern, J. Bacteriol. (1990) 172: 3250–3256 andBartsch et al. J. Bacteriol. (1990) 172: 7035–7042) or S. cerevisiae(André and Jauniaux, Nucl. Acid Res. (1990) 18: 3049).

4hbD gene encoding the 4-hydroxybutyrate dehydrogenase: C. kluyveri(Söhling and Gottschalk, 1996, J. Bacteriol. 178, 871–880).

4-hydroxybutyryl CoA transferase gene: C. aminobutyricum (Willadsen andBuckel, FEMS Microbiol. Lett. (1990) 70: 187–192) or: C. kluyveri(Söhling and Gottschalk, 1996, J. Bacteriol. 178, 871–880).

Other sources of these genes in addition to the listed microorganismswhich are of mammalian or plant origin:

Glutamate dehydrogenase: (Syntichaki et al. (1996) Gene 168: 87–92),maize (Sakakibara et al. (1995), Plant Cell Physiol. 36: 789–797), human(Tzimagiogis et al. (1993), Hum. Genet. 91: 433–438), mouse (Tzimagiogiset al. (1991), Biochem. Biophys. Acta 1089: 250–253), Amuro et al.(1990), Biochem. Biophys. Acta 1049: 216–218).

α-ketoglutarate transaminase: (Park et al. (1993), J. Biol. Chem. 268:7636–7639), Kwon et al. (1992), J. Biol. Chem. 267: 7215–7216), rat(Thakur et al. (1988), Biochem. Int. 16:235–243), rabbit (Kirby et al.(1985), Biochem. J. 230: 481–488).

glutamate decarboxylase: tomato (Gallego et al. (1995), Plant Mol. Biol.27: 1143–1151), human (Bu et al. (1994), Genomics 21:222–228), cat (Chuet al. (1994), Arch. Biochem. Biophys. 313: 287–295), plant (Baum et al.(1993), J. Biol. Chem. 268: 19610–19617).

Regulation of glutamate dehydrogenase expression has been studiedprimarily in E. coli. The corresponding gdhA gene is highly expressed inglucose/ammonia minimal medium and moderately catabolite repressed.Excess glutamate is degraded by aspartate aminotransferase (encoded byaspC). Two REP sequences downstream of the glutamate dehydrogenase geneare involved in mRNA stabilization. The P. fluorescens glutamatedehydrogenase gene shows similar regulation by glucose. Glutamatedehydrogenase from both P. furiosus and C. glutamicum is expressed in E.coli because they complement a gdhA mutation.

The gab gene cluster is only expressed at low constitutive levels due tocatabolite repression by glucose and ammonia. When a poor nitrogensource or succinate as carbon source are supplied the operon isderepressed. Thus, both cAMP/CRP and NtrC regulate the promoter, inaddition to a specific repressor encoded by gabC. The promoter thatregulates gabT is located upstream of gabD. Succinate semialdehydedehydrogenases are encoded by gabD and sad. These activities could bedeleterious for the purpose of P4HB or PHB-4HB production although theirexpression is expected to be repressed by the presence of sufficientglucose and nitrogen sources. Glutamate decarboxylase is a rare enzymeamong the Enterobacteriacea. It is pyridoxal phosphate dependent andwell expressed at low pH.

Pathways to 4-hydroxybutyryl-CoA from Arginine, Putrescine, Glutamineand Proline Via GABA

Bacteria such as Escherichia coli are capable of catabolizing at leastfour different amino acids (arginine, proline, glutamine, and glutamate)to produce GABA, which can be converted as described above to4-hydroxy-butyryl-CoA. These catabolic pathways are depicted in FIG. 3.

E. coli contains at least two activities, encoded by speA and adi, thatcan decarboxylate arginine to agmatine. Putrescine and urea are formedfrom agmatine by the action of agmatine ureohydrolase, encoded by speB.Putrescine donates an amino group to α-ketoglutarate to form4-aminobutyraldehyde and glutamate in a reaction catalyzed by theproduct of the pat gene, putrescine aminotransferase. The4-aminobutyraldehyde is oxidized to GABA by aminobutyraldehydedehydrogenase, encoded by prr. The synthesis of agmatine ureohydrolase,putrescine aminotransferase, and aminobutyraldehyde dehydrogenase isdually controlled by catabolite repression and nitrogen availability.Catabolite repression of agmatine ureohydrolase, but not that ofputrescine aminotransferase or aminobutyraldehyde dehydrogenase, can berelieved by cAMP. Agmatine ureohydrolase synthesis is induced byarginine and agmatine. Arginine decarboxylase synthesis is not sensitiveto catabolite repression or to stimulation by nitrogen limitation orsubject to substrate induction (Shaibe et al., J. Bacteriol. 163:938,1995). There is a second arginine decarboxylase in E. coli which appearsto be specialized for catabolism rather than biosynthesis of arginine,and this protein is encoded by the adi gene (Stim and Bennett, J.Bacteriol. 175:1221, 1993). It is induced under conditions of acidic pH,anaerobiosis, and rich medium.

Proline is degraded in E. coli by the product of the putA gene, whichcatalyzes successive oxidations of proline to pyrroline 5-carboxylateand then to glutamate. The first step is FAD-dependent, and thus thePutA protein is membrane-associated. This same protein also acts as arepressor of the put operon in the absence of proline. The put operon issubject to catabolite repression (McFall and Newman, pp. 358–379, inNeidhardt, ed., Escherichia coli and Salmonella typhimurium: cellularand molecular biology, ASM Press, Washington, D.C., 1996).

Glutamine is converted to glutamate in E. coli by glutamate synthase,the product of the gltB and gltD genes. Two molecules of glutamate areformed by the donation of an amino group by glutamine toα-ketoglutarate. The activity of E. coli glutamate synthase is high whenthis organism is grown in ammonia-containing minimal medium and low whenit is grown in the presence of glutamate or glutamate-generatingnitrogen sources if nitrogen is limiting (Reitzer, pp. 391–407, inNeidhardt, ed., Escherichia coli and Salmonella typhimurium: cellularand molecular biology, ASM Press, Washington, D.C., 1996).

These pathways can be realized for the production ofpoly(4-hydroxybutyrate) in an organism such as E. coli by relying uponthe organism's own genes or by importing such genes from another sourceinto the organism of interest. These genes can be acquired from manyorganisms, such as:

speA encoding arginine decarboxylase: Escherichia coli (Moore and Boyle,J. Bacteriol. 172:4631, 1990), Synechocystis sp. (Kaneko et al., DNARes. 3:109, 1996), Helicobacter pylori (Tomb et al., Nature 388:539,1997), thale cress (Arabidopsis thaliana) (Watson et al., Plant Physiol.114:1569, 1997), soybean (Glycine max) (Nam et al., Plant Cell Physiol.38:1156, 1997), clove pink (Dianthus caryophyllus) (Chang et al., PlantPhysiol. 112:863, 1996), pea (Pisum sativum) (Perez-Amador et al., PlantMol. Biol. 28:997, 1995), tomato (Lycopersicon esculentum) (Rastogi etal., Plant Physiol. 103:829, 1993), oat (Avena sativa) (Bell andMalmberg, Mol. Gen. Genet. 224:431, 1990), plants of the familyBrassicaceae (Barbarea vulgaris, Nasturtium officinale, Arabisdrummondii, Aethionema grandiflora, Capsella bursa-pastoris, Arabidopsisarenosa, Sisymbrium altissimum, Thellungiella salsuginea, Polanisiadodecandra, Stanleya pinnata, Carica papaya, Brassica oleracea, Brassicanigra, Theobroma cacao) (Galloway et al., Mol. Biol. Evol. 15, 1998),rat (Morrissey et al., Kidney Int. 47:1458, 1995).

adi encoding biodegradative arginine decarboxylase: Escherichia coli(Stim and Bennett, J. Bacteriol. 175:1221, 1993).

speB encoding agmatine ureohydrolase: Escherichia coli (Szumanski andBoyle, J. Bacteriol. 172:538, 1990), Streptomyces clavuligerus (Aidoo etal., Gene 147:41, 1994), Bacillus subtilis (Presecan et al.,Microbiology 143:3313, 1997), Synechocystis sp. (Kaneko et al., DNA Res.3:109, 1996), Methanobacterium thermoautotrophicum (Smith et al., J.Bacteriol. 179:7135, 1997), Archaeoglobus fulgidus (Klenk et al., Nature390:364, 1997).

pat encoding putrescine aminotransferase and prr encodingaminobutyraldehyde dehydrogenase: Escherichia coli (Shaibe et al., J.Bacteriol. 163:938, 1985).

gltBD encoding glutamate synthase: Escherichia coli (Oliver et al., Gene60:1, 1987), Aquifex aeolicus (Deckert et al., Nature 392:353, 1998),Azospirillum brasilense (Pelanda et al., J. Biol. Chem. 268:3099, 1993),alfalfa (Medicago sativa) (Gregerson et al., Plant Cell 5:215, 1993),baker's yeast (Saccharomyces cerevisiae) (Filetici et al., Yeast 12:1359, 1996; Cogoni et al., J. Bacteriol. 177:792, 1995), Methanococcusjannaschii (Bult et al., Science 273:1058, 1996), Methanobacteriumthermoautotrophicum (Smith et al., J. Bacteriol. 179:7135, 1997),Bacillus subtilis (Petit et al., Mol. Microbiol. 29:261, 1998),Azospirillum brasilense (Mandal and Ghosh, J. Bacteriol. 175:8024,1993).

putA encoding pyrroline-5-carboxylate reductase: Streptomyces coelicolor(Redenbach et al., Mol. Microbiol. 21:77, 1996), Mycobacteriumtuberculosis (Cole et al., Nature 393:537, 1998), Haemophilus influenzae(Fleischmann et al., Science 269:496, 1995), Escherichia coli (Blattneret al., Science 277:1453, 1997), baker's yeast (Saccharomycescerevisiae) (Science 265:2077, 1994), Vibrio alginolyticus (Nakamura etal., Biochim. Biophys. Acta 1277:201, 1996), Pseudomonas aeruginosa(Savoiz et al., Gene 86:107, 1990), Klebsiella pneumoniae (Chen andMaloy, J. Bacteriol. 173:783, 1991), Salmonella typhimurium (Allen etal., Nucleic Acids Res. 21:1676, 1993), Agrobacterium tumefaciens (Choet al., J. Bacteriol. 178:1872, 1996), Sinorhizobium meliloti(Jimenez-Zurdo et al., Mol. Microbiol. 23:85, 1997), Rhodobactercapsulalus (Keuntje et al., J. Bacteriol. 177:6432, 1995),Bradyrhizobium japonicum (Straub et al., Appl. Environ. Microbiol.62:221, 1996), Synechocystis sp. (Kaneko et al., DNA Res. 3: 109, 1996),Shewanella sp. (Kato et al., J. Biochem. 120:301, 1996), Pholobacteriumleiognalhi (Lin et al., Biochem. Biophys. Res. Commun. 219:868, 1996),Helicobacter pylori (Tomb et al., Nature 388:539, 1997), cultivatedmushroom (Agaricus bisporus) (Schaap et al., Appl. Environ. Microbiol.63:57, 1997), soybean (Glycine max) (Delauney and Verma, Mol. Gen.Genet. 221:299, 1990), human (Homo sapiens) (Campbell et al., Hum.Genet. 101:69, 1997).

The arginine, proline, glutamine, or glutamate can be suppliedexogenously to the poly(4-hydroxybutyrate)-producing organism, or it canbe synthesized in the host from another carbon source, preferably aninexpensive one such as glucose. E. coli, for example, synthesizes allof these compounds from glucose, but generally only to an extentsufficient for growth.

Strains of E. coli that overproduce these compounds have been developed.Tujimoto et al. (U.S. Pat. No. 5,378,616) describe an E. coli mutantthat accumulates glutamate. Momose et al. (U.S. Pat. No. 4,430,430)describe the overexpression of the argA gene in E. coli, which leads toarginine accumulation. Proline-resistant mutants of E. coli thatoverexpress proline synthesis genes can accumulate proline (Wang et al.,Chin. J. Biotechnol. 6:27, 1990). Tobacco plants which overexpressbacterial proline synthesis genes were also shown to accumulate proline(Sokhansandzh et al., Genetika 33:906, 1997). Furthermore, E. coli andother bacteria accumulate glutamate, GABA, and proline as a response tohigh medium osmolarity (McLaggan et al., J. Biol. Chem. 269:1911, 1994;Measures, J. C., Nature 257:398, 1975; Schleyer et al., Arch. Microbiol.160:424, 1993; Botsford et al., Appl. Environ. Microbiol. 60:2568,1994).

Pathway to 4-hydroxybutyryl CoA from Succinate

The complete biochemical pathway for the conversion of succinate to4HB-CoA (FIG. 4) has been characterized in Clostridium kluyveri (Söhlingand Gottschalk, 1993, Eur. J. Biochem. 212, 121–127; Wolffet al., 1993,Appl. Environ. Microbiol. 59, 1876–1882; Scherf et al., 1994, Arch.

Microbiol. 161, 239–245). More recently, the genes encoding the C.kluyveri succinyl-CoA: CoA transferase (cat1), succinate-semialdehydedehydrogenase (sucD) and 4-hydroxybutyrate dehydrogenase (4hbD) havebeen identified (Söhling and Gottschalk, 1996, J. Bacteriol. 178,871–880). These genes are located in a contiguous stretch of DNA on theC. kluyveri chromosome and flanked by three genes of unknown function(orfZ, orfY and sigL). The genes appear to be induced by succinate inthe growth medium. The gene encoding 4-hydroxybutyryl CoA transferasewas not identified in these studies.

Identification of Alternative Genes Encoding Enzymes that Operate in theSynthesis of 4-hydroxybutyrate

Alternative genes encoding enzymes that operate in the conversion ofeither α-ketoglutarate or succinate to 4HB can be isolated bycomplementation or expression studies: glutamate-succinic semialdehydetransaminase genes can be isolated from gene libraries because of theability of this gene to complement an E. coli gabT mutation forutilization of γ-aminobutyric acid as nitrogen source. Likewise,mutations in glutamate dehydrogenase and glutamate decarboxylase genesin E. coli can be complemented. Expression of alternative4-hydroxybutyrate dehydrogenase genes will allow E. coli to utilize4-hydroxybutyrate as a carbon source. Enzyme homology searches using theBLASTP program and the GenBank database suggest the presence of4-hydroxybutyrate dehydrogenase homologs in the E. coli genome. Theseproteins have been identified with the genetic index numbers: gi |1788795 and gi | 1790015.

Importance of Integration; Screening for Polymer Production

It is important for efficient PHA production that strains do not losethe capability to synthesize the biopolymer for the duration of theinoculum train and the production run. Loss of any of the phb genesresults in loss of product whereas loss of any of the genes that providenew monomers results in heterogeneous product formation. Both areundesirable and stable propagation of the strain is therefore required.Unfortunately, merely integrating the gene encoding the transferase orsynthase does not result in significant polymer production. It isnecessary to enhance enzyme expression, through alteration of thepromoter region or mutagenesis or other known techniques, followed byscreening for polymer production. Using these techniques, integration ofthe genes in the strains described in the examples was determined to bestable for at least 50 generations, sufficient for production in 100,000L vessels.

Growth and morphology of these recombinant PHA producers is notcompromised by the presence of phb genes on the chromosome. During theselection procedures, individual integrants are selected on minimalmedium plates circumventing the isolation of auxotrophic strains. Growthrates of the different phb integrants were similar to that of thewild-type E. coli strains from which the PHB producers were derived. Theaddition of the phb genes to the E. coli chromosome did not affect thedownstream processing of these strains, as they were still easily lysedby conventional methods.

The present invention will be further understood by reference to thefollowing non-limiting examples.

EXAMPLE 1 Minimal Requirements for PHB-4HB Synthesis

It has been previously shown that the minimum requirements for thesynthesis of poly-(R-3-hydroxybutyrate) (PHB) are the purified PHAsynthase from A. eutrophus and the substrate (R)-3-hydroxybutyryl-CoA.4-Hydroxybutyryl-CoA can be prepared in situ from acetyl-CoA and4-hydroxybutyrate via a transthioesterification reaction catalyzed bythe enzyme 4-hydroxybutyryl-CoA transferase, isolated from Clostridiumaminobutyricum. This enzyme will also catalyze the formation of(R)-3-hydroxybutyryl-CoA from the free acid and acetyl-CoA. Thus theminimum requirements for the in situ synthesis of 4-hydroxybutyryl-CoAand its co-polymerization with (R)-3-hydroxybutyryl-CoA to formP(3HB-co-4HB) would include PHA synthase, (R)-3-hydroxybutyric acid,4-hydroxybutyric acid, acetyl-CoA and 4-hydroxybutyryl-CoA transferasein a buffered aqueous solution. This was demonstrated as follows:

To potassium phosphate buffer (1 ml, 100 mM, pH 7.5) the following wereadded:

-   -   acetyl-CoA (0.5 mL, 30 mM)    -   4-hydoxybutyric acid sodium salt (50 μl, 2 M)    -   (R)-3-hydroxybutyric acid sodium salt (100 μl, 1 M)    -   4-hydroxybutyryl-CoA transferase (10 mg, 25 units)    -   PHA synthase (0.05 mg)        The reaction was allowed to stand at room temperature overnight.        The formation of insoluble PHA granules was noted. Insoluble        material was pelleted by centrifugation and freeze dried (0.65        mg). This material had a sticky consistency. Organic material        was extracted with CDCl₃ and analyzed by ¹H-NMR. NMR analysis        confirmed the formation of        poly-((R)-3-hydroxybutyrate-co-4-hydroxybutyrate) containing        approximately 20% 4-hydroxybutyric acid. The NMR spectrum        matches a literature spectrum of        poly-((R)-3-hydroxybutyrate-co-4-hydroxybutyrate) (Doi, Y. et        al., Macromolecules 1988, 21: 2722–2727).

EXAMPLE 2 Poly(4-hydroxybutyrate) (P4HB) Synthesis in E. coli Using aPlasmid Encoded Pathway

The hbcT gene from C. kluyveri was expressed in E. coli using standardmolecular biological techniques. The gene is placed in an appropriatevector behind a strong promoter and under conditions that driveexpression from this promoter. 4HBCT is produced.

Strains of E. coli were equipped with plasmid pFS30 which contains thegenes encoding 4-hydroxybutyryl-CoA transferase from C. kluyveri and PHBsynthase from R. eutropha. Theses genes are expected to convert4-hydroxybutyric acid into 4-hydroxybutyryl-CoA which is subsequentlypolymerized to poly(4-hydroxybutyrate). Strains were grown in 250 mlErlenmeyer flasks containing 50 to 100 ml 10% LB liquid medium with4-hydroxybutyrate, alone or in combination with glucose, as carbonsource. Cultures were incubated at 30 to 33° C. with shaking at 150 or200 rpm. Cultures were harvested after 24 hours of incubation andanalyzed for PHA. E. coli MBX1177 (a spontaneous mutant of strain DH5αselected for growth on minimal 4-HB medium) with pFS30 accumulates 67%of its cell dry weight as a P4HB homopolymer:

host volume rpm 4HB glc T % LB % PHA F(4HB) 19  50 ml 150 5 2 33 10 <51.0 184 100 ml 150 5 2 33 10 38.9 1.0 816 100 ml 200 5 0 32 1019.3 >0.99 817 100 ml 200 5 0 32 10 12.8 >0.99 821 100 ml 200 5 0 32 1024.8 >0.99 1177  50 ml 150 5 0 33 10 14.8 1.0 1177 100 ml 200 5 2 30 1067.1 1.0

EXAMPLE 3 Poly(4-hydroxybutyrate) (P4HB) Synthesis in E. coli Using aPlasmid Encoded PHA Synthase

Strains of E. coli were equipped with plasmid pFS16, which contains thegene encoding 4-hydroxybutyryl-CoA transferase from C. kluyveri. Thisgene is expected to convert 4-hydroxybutyric acid into4-hydroxybutyryl-CoA which is subsequently polymerized by achromosomally encoded PHB synthase into P4HB. Strains were grown in 250ml Erlenmeyer flasks containing 50 to 100 ml 10% LB or 100% LB liquidmedium with 4-hydroxybutyrate, alone or in combination with glucose, ascarbon source. Cultures were incubated at 32 to 37° C. with shaking at 0to 250 rpm. Cultures were harvested after 24 hours of incubation andanalyzed for PHA. E. coli MBX769 with pFS16 accumulates 67% of its celldry weight as a P4HB homopolymer. Formation of 4HB containing PHAs isconsequently not dependent on a plasmid encoded PHB synthase.

host volume rpm 4HB glc T % LB % PHA F(4HB) 777  50 ml 250 5 0 37 1007.6 0.36 769  50 ml 250 5 0 37 100 0 — 769  50 ml 100 5 0 33 10 8.0 0.18769 100 ml 150 5 2 33 10 16.4 0.25 769 100 ml 200 5 2 32 10 43.5 0.37769 100 ml 0 5 0 33 10 13.6 0.29 769 100 ml 0 5 0 33 10 19.8 0.32 769100 ml 250 5 0 37 10 2.4 0.002

EXAMPLE 4 Construction of Plasmids for Chromosomal Integration of phbGenes

Plasmid pMUXC₅cat contains the phbC gene from Z. ramigera on atransposable element for integration of this gene on the chromosome of arecipient strain (FIG. 5). Strong translational sequences were obtainedfrom pKPS4 which encodes PHA synthase encoding phaC1 from P. oleovoransin the pTrc vector (Pharmacia). In this construct, phaC1 is preceded bya strong ribosome binding site: AGGAGGTTTTT(-ATG) (SEQ ID NO:4). ThephaC1 gene, including the upstream sequences, was cloned as a bluntended EcoRI-HindIII fragment in the SmaI site of pUC18Sfi to givepMSXC₃. A blunt ended cat gene cassette was subsequently cloned in theblunt-ended Sse8387II site, resulting in pMSXC₃cat. At this point, allof the phaC1 coding region except the 5′ 27 base pairs were removed as aPstI-BamHI fragment and replaced by the corresponding fragment from thephbC gene from Z. ramigera. The resulting plasmid, pMSXC₅cat, encodes ahybrid PHB synthase enzyme with the 9 amino terminal residues derivedfrom the P. oleovorans PHA synthase and the remainder from Z. ramigera.The C₅cat cassette was then excised as an AvrII fragment and cloned inthe corresponding sites of pUTHg, thereby deleting the mercuryresistance marker from this vector. The resulting plasmid, pMUXC₅cat,contains a C₅cat mini-transposon in which phbC is not preceded by apromoter sequence. Expression of the cassette upon integration istherefore dependent on transcriptional sequences that are provided bythe DNA adjacent to the integration site.

pMSXTp₁AB₅kan2 was constructed from pMSXTp₁kan as follows (FIG. 6).First pMSXTp₁kan was digested with NdeI, filled in with Klenow andreligated to obtain pMSXTp₁kan2 in which the NdeI site is deleted. Thisdeletion results in a unique NdeI site just upstream of phbA of Z.ramigera during later stages of the cloning procedure.

B₅ was cloned as a NarI fragment from pZT1 and cloned in the HincII siteof pUC18Sfi to generate pMSXB₅. A₅ was inserted as an FseI/blunt-SalIfragment in the Ecl136II-SalI sites resulting in pMSXAB₅ andregenerating the Z. ramigera AB₅ intergenic region. pMSXAB₅cat wascreated by inserting a promoterless cat cassette in the HindIII site ofpMSXAB₅. The AB₅ fragment from pMSXAB₅cat was cloned as a EcoRI-PstIfragment into the SmaI site of pMSXTp₁kan2 giving pMSXTp₁AB₅kan2.

Expression of phbAB₅ was improved by introduction of a strong promoterupstream of these genes (FIG. 6). This promoter was generated with setsof oligonucleotides that provide upstream activating sequences, a −35promoter region, a −10 promoter region with transcriptional startsite(s), and mRNA sequences with possible stabilizing functions. PlasmidpMSXTp₁AB₅kan2 was digested with PstI/XbaI and a fragment containing the−10 region of the lac promoter was inserted as a fragment obtained afterannealing oligonucleo-tides

3A (5′GGCTCGTATAATGTGTGGAGGGAGAACCGCCGGGCTCGCGCCGTT) (SEQ ID NO:5) and3B (5′ CTAGAACGGCGCGAGCCCGGCGGTTCTCCCTCCACA CATTATACGAGCCTGCA) (SEQ IDNO:6).

Next, a fragment containing a consensus E. coli pho box and −35 promoterregion were inserted into the PstI site as a fragment obtained afterannealing the oligonucleotides: 2A: (5′ TCCCCTGTCATAAAGTTGTCACTGCA) (SEQID NO:7) and 2B (5′ GTGACAACTTTATGACAGGGGATGCA) (SEQ ID NO:8). Next, themessenger stabilizing sequence including the transcriptional start sitefrom AB₅ was inserted into the XbaI-NdeI sites as a fragment obtainedafter annealing the oligonucleotides: 4A (5′: CTAGTGCCGGACCCGGTTCCAAGGCCGGCCGCAAGGCTGCCAGAACTGAGGAAGCACA) (SEQ ID NO:9) and 4B:(5′TATGTGCTTCCTCAGTTCTGGCAGCCTTGCGGCCGGCCTTGGAACCGGGTCCGGCA) (SEQ IDNO:10). The resulting plasmid is pMSXp₁₂AB₅kan2. The AvrII fragment,containing Tp₁₂AB₅kan2 was cloned into pUTHg cut with AvrII and used forintegration into the genome of MBX379 and MBX245.

The p12AB₅kan expression cassette were then excised as a 2.8 kb AvrIIfragment and ligated into the AvrII site of pUTHg and transformed intoE. coli strain CC118 λpir to obtain plasmids pMUXp₁₂AB₅kan. This plasmidwas then transformed into E. coli S17-1 λpir and used to insertp12AB₅kan expression cassettes into the chromosome of E. coli strains byconjugation (Herrero et al. J. Bacteriol. 1990, 172: 6557–6567).

EXAMPLE 5 Integration of phb Genes into the Chromosome of E. coli

Material and Methods

E. coli strains were grown in Luria-Bertani medium (Sambrook et. al.,Molecular Cloning, a laboratory manual, 2nd Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.) at 37° C. or 30° C. or inminimal E2 medium (Lageveen el al., Appl. Environ. Microbiol. 1988, 54:2924–2932). DNA manipulations were performed on plasmid and chromosomalDNA purified with the Qiagen plasmid preparation or Qiagen chromosomalDNA preparation kits according to manufacturers recommendations. DNA wasdigested using restriction enzymes (New England Biolabs, Beverly, Mass.)according to manufacturers recommendations. DNA fragments were isolatedfrom 0.7% agarose-Tris/acetate/EDTA gels using a Qiagen kit.

Plasmid DNA was introduced into E. coli cells by transformation orelectroporation (Sambrook et al. Molecular Cloning, a laboratory manual,2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).Transposition of phb genes from the pUT vectors was achieved by matingof the plasmid donor strain and the recipient (Herrero et al. J.Bacteriol. (1990) 172: 6557). The recipient strains used werespontaneous naladixic acid or rifampicin resistant mutants of E. coliderived from either LS5218 or MBX23. MBX23 is LJ14 rpoS::Tn10 in whichthe rpoS::Tn10 allele was introduced by P1 transduction from strain 1106(Eisenstark). Recipients in which phb genes have been integrated intothe chromosome were selected on naladixic acid or rifampicin platessupplemented with the antibiotic resistance specified by themini-transposon, kanamycin or chloramphenicol. Oligonucleotides werepurchased from Biosynthesis or Genesys. DNA sequences were determined byautomated sequencing using a Perkin-Elmer ABI 373A sequencing machine.DNA was amplified using the synthase-chain-reaction in 50 microlitervolume using PCR-mix from Gibco-BRL (Gaithersburg, Md.) and an EricompDNA amplifying machine.

Accumulated PHA was determined by gas chromatographic (GC) analysis asfollows. About 20 mg of lyophilized cell mass was subjected tosimultaneous extraction and butanolysis at 110° C. for 3 hours in 2 mLof a mixture containing (by volume) 90% 1-butanol and 10% concentratedhydrochloric acid, with 2 mg/mL benzoic acid added as an internalstandard. The water-soluble components of the resulting mixture wereremoved by extraction with 3 mL water. The organic phase (1 μL at asplit ratio of 1:50 at an overall flow rate of 2 mL/min) was analyzed onan HP 5890 GC with FID detector (Hewlett-Packard Co, Palo Alto, Calif.)using an SPB-1 fused silica capillary GC column (30 m; 0.32 mm ID; 0.25μm film; Supelco; Bellefonte, Pa.) with the following temperatureprofile: 80° C., 2 min; 10° C. per min to 250° C.; 250° C., 2 min. Thestandard used to test for the presence of 4-hydroxybutyrate units in thepolymer was γ-butyrolactone, which, like poly(4-hydroxybutyrate), formsn-butyl 4-hydroxybutyrate upon butanolysis. The standard used to testfor 3-hydroxybutyrate units in the polymer was purified PHB.

1-Methyl-3-nitro-1-nitroso-guanidine (NTG) mutagenesis was performed asdescribed by Miller (A short course in bacterial genetics, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.) using a 90 minutetreatment with 1 mg/ml NTG corresponding to 99% killing.

Results

C₅cat was introduced into the chromosome of MBX23 by conjugation usingS17-1 λpir (pMUXC₅cat) the donor strain. The conjugation mixture wasspread on LB/Nl/Cm plates and integrants were obtained of which 40% weresensitive to ampicillin, indicating that no plasmid was present in thesestrains. Five integrants were transformed with pMSXAB₅cat (Ap^(r)) andgrown on LB/Ap/Cm/2% glucose to examine biosynthetic activity of PHBsynthase. MBX326 expressed the highest synthase activity and was used infurther studies. Expression of PHB synthase was increased by restreakingMBX326 successively on LB plates containing 100, 200, 500 and 1000 μg/mlchloroamphenicol. Strain MBX379 is derived from MBX326 and exhibitschloramphenicol resitence up to 1000 μg/ml.

E. coli S17-1 λpir containing pMUXp₁₂AB₅kan was mated with MBX379.Transgenic strains in which phbAB₅kan had integrated on the chromosomewere selected on LB/Nl/Km plates. Among the integrants, PHB producerswere identified on LB/glucose plates and MBX677 (MBX379:: p₁₂AB₅kan) wasused for further studies. The PHB level in this strain grown inLuria-Bertani/2% glucose medium was 58% whereas 38% PHB was accumulatedin minimal medium supplemented with 2% glucose.

EXAMPLE 6 Mutagenesis of Transgenic E. coli Strains for Enhanced PHBProduction

Mutagenesis using NTG or EMS was used to improve PHB formation inMBX680. Strain MBX769 and MBX777 were selected after treatment of MBX680with EMS and NTG respectively. These strains are able to grow onR2-medium supplied with 1% glucose, 0.5% corn steep liquor and 1 mg/mlchloroamphenicol. MBX769 was grown in 50 ml R-10 medium/0.5% CSL with 2or 3% glucose at 37° C. for 20 to 26 hours. PHB was accumulated to 71%of the cell dry weight. Similarly, MBX769 was grown in 50 ml LB with orwithout 0.375 g/L KH₂PO₄, 0.875 K₂HPO₄ and 0.25 (NH₄)₂SO₄ and a total of50 g/L glucose (five aliquots were added over the course of theincubation). After 63 hours of incubation, PHB had accumulated up to 96%of the cell dry weight. PHB levels in MBX777 strain grown inLuria-Bertani/2% glucose medium was 67% whereas in minimal mediumsupplemented with 2% glucose 57% PHB was accumulated.

Improved transgenic E. coli strains with a chromosomal phbC gene wereobtained by P1 transduction of the C5cat allele from MBX379 into LS5218,LS5218 fadAB₁₀₁::Tn10 and LS5218 fadR⁺ zcf117::Tn10. The resultingstrains are MBX816, MBX817 and MBX821, respectively.

EXAMPLE 7 Poly(4-hydroxybutyrate) (P4HB) Synthesis in E. coli Using anEndogenous 4-hydroxybutyryl-CoA Transferase Activity

E. coli contains an endogenous gene encoding an enzyme with4-hydroxybutyryl-CoA transferase activity. Strains MBX821 and 1231 weregrown in 250 ml Erlenmeyer flasks containing 50 to 100 ml 10% LB liquidmedium with 4-hydroxybutyrate, alone or in combination with glucose, ascarbon source. MBX1231 is a mutant of MBX821 obtained after treatmentwith 1-methyl-3-nitro-1-nitrosoguanidine and selected on platescontaining 500 μg/ml chloramphenicol. Cultures were incubated at 32 to33° C. with shaking at 200 rpm. Cultures were harvested after 24 hoursof incubation and analyzed for PHA. Table x shows that these strainsaccumulate 2.5 to 3.5% of the cell dry weight as a P4HB homopolymer.P4HB formation in this strain is not dependent on a plasmid encoded PHBsynthase nor a heterologously expressed 4-hydroxybutyryl-CoAtransferase. When these strains are grown on solid media, P4HB levelsare improved to around 11%.

host volume rpm 4HB glc T % LB % PHA F(4HB)  821 100 200 5 2 32 10  2.51.0 1231 100 200 5 2 33 10  3.5 1.0  821 on plate 5 2 RT 10 10.5 1.01231 on plate 5 2 RT 10 11.5 1.0

EXAMPLE 8 A Screening Method for Air Insensitive 4-hydroxybutyryl CoATransferase

The 4-hydroxybutyryl-CoA transferase from C. kluyveri appears to beinhibited by air, most likely by oxygen. Oxygen insensitive mutants canbe screened for by growing mutants of an E. coli strain that harbors the4-hydroxybutyryl-CoA transferase encoding hbcT gene on a plasmid and aPHA synthase gene on the chromosome, for P4HB synthesis under highoxygenation conditions and searching for white colonies (indicative ofPHA accumulation) where the majority of the population forms greycolonies. Oxygen insensitive strains, MBX240 [pFS16], MBX379 [pFS16] andMBX830 [pFS16], were identified using this method. Populations ofmutants can be generated in vivo by treating the original strain withchemical mutagens such as N-methyl-N′-nitro-N-nitrosoguanidine orethylmethanesulfonate or with ultraviolet radiation. Alternatively, anhbcT containing plasmid can be mutagenized in vitro with hydroxylamine.Mutants expressing a functional 4-hydroxybutyryl-CoA transferase arethen screened for on solid media or highly oxygenated liquid media forP4HB formation from 4-hydroxybutyrate.

EXAMPLE 9 A Screening Method for Additional E. coli Genes Encoding4-hydroxybutyryl CoA Biosynthetic Enzymes

Expression of the enzymatic activity that converts 4HB to 4HB-CoA inMBX821 or 1231 may be elevated by mutagenesis. Appearance of P4HB inMBX821 and 1231 grown on solid media took approximately 150 hours.Mutants with improved P4HB accumulation characteristics can be screenedfor after random mutagenesis of these strains with chemical mutagenssuch as N-methyl-N′-nitro-N-nitrosoguanidine or ethylmethanesulfonate orwith ultraviolet radiation. Desired mutants form white colonies within 2to 5 days of incubation in the presence of 4-hydroxybutyrate.

EXAMPLE 10 A Screening Method for Other Genes Encoding 4-hydroxybutyrylCoA Biosynthetic Enzymes

Because applications involving plant systems require DNA with a high GCcontent, alternative 4-hydroxybutyryl CoA biosynthetic genes need to beidentified and isolated. The low GC content of the hbcT gene would makesit a useful probe for identification and isolation of homologous genesfrom other AT-rich DNA containing microorganisms. HbcT genes with a highGC content however will not be identified by this method. E. colistrains that have a chromosomally integrated phbC gene encoding PHAsynthase can be used to screen for such genes. For applications wheregenes are introduced into plants it is desirable to use DNA with a highGC content (Perlak F. J. et al., Proc. Natl. Acad. Sci. USA (1991) 88:3324). When hbcT genes are expressed in E. coli MBX379 for instance,this strain is able to produce a P4HB polymer on agar plates containing4-hydroxybutyrate in addition to the common nutrients. The formation ofP4HB gives the colony an easily distinguishable white phenotype. Thus,gene libraries of PHB-co-4HB producing organisms. such as R. eutropha,A. latus, P. acidovorans, C. testosteroni and others are introduced intoMBX379 or similar strains and directly plated on 4HB containing growthmedium. White colonies are selected and the composition of theaccumulated PHA is determined. Gene libraries are readily constructedfrom organisms of choice by isolating genomic DNA and cloning arepresentative collection of DNA fragments in plasmid vectors.Representative libraries should have 5,000 to 100,000 individualcolonies. Libraries are either made as a broad host range library invectors such as pLAFR3 or as E. coli libraries in vectors such as pUC19, pBR322. Depending on the type of library and the method ofintroducing the library in the host of choice, the genomic DNA fragmentsare either large (17–30 kb) or relatively small (2–6 kb). Libraries areintroduced into the screening strains by electroporation, transformationor conjugation, dependent on the host and the vector used.

In addition to alternative 4-hydroxybutyryl CoA transferases, acyl CoAsynthetases able to utilize 4-hydroxybutyrate as a substrate will beisolated by this method. Examples of genes encoding enzymes with suchgeneral activities are fadD, involved in uptake of long-side chain fattyacids, atoDA, involved in uptake of acetoacetate and short side chainfatty acids, catE, involved in degradation of aromatics, aceAB, encodingsuccinyl CoA synthetase, acsA and acsB encoding acetyl CoA synthetasesand homologs of such genes. Alternatively the substrate specificity ofthese enzymes may be expanded to include 4-hydroxybutyrate byintroducing plasmids with randomly mutagenized acyl CoA synthetase ortransferase genes. Alternatively, the ygfH gene from E. coli whichshares significant homology with the hbcT gene from C. kluyveri may beexplored for 4-hydroxybutyryl CoA activity.

EXAMPLE 11 Endogenous Synthesis of 4HB-CoA from α-ketoglutarate

α-Ketoglutarate is a cellular metabolite that can be converted to 4HB asshown in FIG. 7. The pathway consists of a cyclic reaction catalyzed bythe gabT, gadA/gadB and gdhA gene products. Formation of succinic acidsemialdehyde from this cycle is favored once the product is furtherconverted to 4HB-CoA by 4-HB dehydrogenase and 4HB-CoA transferase, andpolymerized into a PHA by PHA synthase.

For this purpose the following plasmids were constructed in pMSXcat:

1. pMSX-TD hbcT-4hbD 2. pMSX-ABT gdhA-gadB-gabT 3. pMTX-DBTT4hbD-gadB-gabT-hbcT 4. PMSX-ABTTD gdhA-gadA-gabT-hbcT-4hbD1. 4hbD was obtained from pCK3 by PCR using the primers:

(SEQ ID NO:11) 4HBD-N: 5′CTCTGAATTCAAGGAGGAAAAAATATGAAGTTAT        TAAAATTGGC (EcoRI) (SEQ ID NO:12) 4HBD-C:5′TTTCTCTGAGCTCGGGATATTTAATGATTGTAGG        (SacI).The PCR product was cloned into pCR2.1 (pMBX-D). hbcT was cloned as anSspI-EcoRI fragment from pCK3 and cloned in EcoRV/EcoRI digested pMBX-Dto give pMBX-TD. The artificial hbcT-4hbD operon was excised frompMBX-TD as a NotI-KpnI fragment and ligated into these sites in pUC18Sfior pMSX-TP1 (pMSX-TD and pMSX-TP₁TD respectively) (FIG. 8). The TD orTP₁-TD fragment was excised as a AvrII fragment and ligated into AvrIIdigested pUTkan (pMUX-TD and pMUX-TP₁-TD). This plasmid allows randominsertion of the TD/TP1-TD construct in the chromosome of E. coli.Expression of integrated TD is driven by an endogenous promoter whereasexpression of integrated TP₁-TD is driven by P₁. Recombinants in whichthe construct had integrated were selected lor their ability to grow on4-hydroxybutyrate as sole carbon source. No antibiotic resistance markerwas required to select the desired insertions.

Other genes encoding enzymes that facilitate conversion of succinicsemialdehyde to 4-hydroxybutyryl CoA can be isolated routinely bycomplementation. After introduction of 4hbD homologs such genes conferon wild-type E. coli strains the ability to use 4HB as sole carbonsource.

-   2. An operon consisting of gdhA-gadA-gabT was created in plasmid    pUC18Sfi and inserted in the E. coli chromosome using the pUTkan    vector. Recipients of the construct were isolated on    E2/glycerol/_γ-hydroxybutyrate/N1plates. Because the recipient    strain is unable to use γ-hydroxybutyrate as nitrogen source (due to    a gabT mutation), only those strains that express the operon grow on    this medium.

The gdhA gene was obtained from the E. coli chromosome using PCR and thefollowing primers:

(SEQ ID NO:13) GH-Up: 5′ AACGAATTCAATTCAGGAGGTTTTTATGGATCAGACATATTCTCTGGAGTC (EcoRI) (SEQ ID NO:14) GH-Dn: 5′TTGGGAGCTCTACAGTAAGAAATGCCGTTGG (SacI).The gadB gene was obtained from the E. coli chromosome using PCR and thefollowing primers:

(SEQ ID NO:15) GB-Up: 5′ TAAGAGCTCAATTCAGGAGGTTTTTATGGATAAGAAGCAAGTAACGGATTTAAGG (SacI) (SEQ ID NO:16) GB-Dn: 5′TTCCCGGGTTATCAGGTATGCTTGAAGCTGTTCTGT TGGGC (XmaI).The gabT gene was obtained from the E. coli chromosome using PCR and thefollowing primers:

(SEQ ID NO:17) GT-Up: 5′ TCCGGATCCAATTCAGGAGGTTTTTATGAACAGCAATAAAGAGTTAATGCAG (BamHI) (SEQ ID NO:18) GT-Dn: 5′GATTCTAGATAGGAGCGGCGCTACTGCTTCGCC (XbaI).

DNA sequence information used to design the above primers was fromGenBank, accession numbers: K02499 (gdhA), M84025 and X71917 (gadB),M88334 (gabT).

The three PCR products were digested with the indicated enzymes andsequentially cloned in the pUC18Sfi vector (pMSX-ABT) (FIG. 9). Theoperon was excised as an EcoRI-SalI fragment and cloned in pMSXTP₁(pMSX-TP₁-ABT). Either the ABT or TP₁-ABT insert was moved to pUTkan toallow insertion of the gdhA-gadA-gabT operon in the chromosome of a gabTmutant of E. coli MBX245. Successful insertions were selected onE2/glycerol/γ-hydroxybutyrate/N1 plates.

Because gabT expression allows the use of γ-hydroxybutyrate as nitrogensource, genes that express this function can be easily selected for onminimal medium plates in which γ-hydroxybutyrate serves as the onlynitrogen source. Expression of gabT at the end of the operonnecessitates the transcription of the upstream genes for which no directselection is available.

Glutamate dehydrogenase functions in this pathway as a source to provideglutamate in catalytic amounts. If sufficient glutamate is present,additional GdhA activity may not be required and incorporation of thisgene in the described constructs is therefore optional.

3. The operons described under 1 and 2 were combined as follows: pMSX-TDwas digested with KpnI, T4 polymerase treated and digested with XhoI;pMSX-ABT or pMSX-BT were digested with HindIII, Klenow treated anddigested with SalI; the purified TD fragment was subsequently ligatedinto the prepared pMSX-ABT and pMSX-BT plasmids (FIG. 9).

EXAMPLE 12 Endogenous Synthesis of 4HBCoA from GABA Precursors

The common metabolite GABA is derived from glutamate and is normallymetabolized via succinic semialdehyde to succinate in centralmetabolism. It may be desirable to improve the pathways to GABA toachieve high levels of the intermediates for P4HB formation. Besides thedirect conversion of α-ketoglutarate to glutamate by glutamatedehydrogenase, this conversion is also part of many transaminationreactions for instance with substrates such as glutamine and other aminoacids, or putrescine. Recombinant and mutant organisms that overproducearginine (the precursor of putrescine), glutamine or proline,consequently have increased levels of glutamate and GABA which can beshunted to 4HB-CoA with gabT, 4hbD and hbcT as described above (FIG.10).

EXAMPLE 13 Endogenous Synthesis of 4HBCoA from Succinate

HbcT is not required for E. coli to grow on 4-hydroxybutyrate when cat1,4hbD and sucD are introduced (Söhling and Gottschalk, 1996, J.Bacteriol. 178, 871–880) possibly because the reverse action of SucD,4HBD and Cat1 converts 4HB to succinate, a central metabolite in E.coli. In principle, these genes together allow the conversion ofsuccinate to 4-HB. The pathway as depicted in FIG. 4 can then beassembled from the cat1, sucD, 4hbD and hbcT genes of C. kluyveri.Alternatively, these genes can be isolated from other Clostridiumspecies such as C. aminobulyricum. Although E. coli does have asuccinyl-CoA:CoA transferase itself (sucCD; Mat-Jan et al. Mol. Gen.Genet. (1989) 215: 276–280), it is desirable to introduce this gene fromanother source because this activity is not prominent in E. coli(Amarasingham and Davis, J. Biol. Chem. (1965) 240: 3664–3668).Alternatively, expression of the E. coli gene can be optimized for thecurrent application.

An operon was constructed for integration in the E. coli chromosomeconsisting of hbcT-cat1-sucD-4hbD. Strains in which integration wassuccessful are able to grown on 4HB if 4hbD is expressed (Sohling andGottschalk, 1996, J. Bacteriol. 178, 871–880). The construction of thisoperon proceeded as follows (FIG. 11):

A BamHI-PstI fragment from pCK3 containing orfY, cat1, sucD and the 5′end of 4hbD was ligated in the corresponding sites of pMSXcat(pMSX-Y1D). The 4hbD gene was completed by inserting the PstI-SacIfragment of pMSX-D in PstI-SphI digested pMSX-Y1D (pMSX-Y1DD). Toachieve this, both fragments in this ligation were T4 polymerase treatedafter the SphI and SacI digestions to create blunt ends before anadditional PstI digestion was started. OrfY in pMSX-Y1DD was replacedwith hbcT by digesting pMSX-Y1DD with BamHI and PacI, followed by bluntending the fragment with Klenow/T4 polymerase and dephosphorylation, andthen ligation of the SspI/EcoRI, Klenow treated hbcT fragment into thisvector (pMSX-T1DD). A fragment providing the regulatory sequences,terminator and promoter was inserted as a blunt ended fragment in theSmaI site of pMSX-T1DD. An integration plasmid for this operon wasconstructed by cloning the insert of pMSX-T1DD as an SfiI fragment intopUTkan.

EXAMPLE 14 Improved Endogenous Synthesis of 4HBCoA

In order to prevent drainage of intermediates from these new pathways,it may be desirable to inactivate the genes encoding aspartatetransaminase (aspC) and the NADP and AND dependent succinic semialdehydedehydrogenases (sad and gabD). Mutations in the individual genes wereobtained from different sources: A strain containing the aspC13Imutation is obtained from the E. coli Genetic Stock Center as strainCGSC5799. The aspC gene maps to minute 21.1 and is therefore linked tothe Tn10 (Tc) marker in CAG12094 (zcc-282 at 22.25 minutes) or CAG18478(zbj-1230 at 20.00 minutes) and to the Tn10 Km marker in CAG12130(zcb-3111 at minute 21.00). No mutations in the gabD gene are known anddeletion of this activity can be achieved by cloning the gene by PCR,insertion of a genetic marker such as antibiotic resistance, integrationusing recBC strains or vectors constructed for this purpose such aspMAK705 and finally, bacteriophage P1 transduction to transfer the geneto the desired host.

EXAMPLE 15 Expression of a PHA Synthase and 4-hydroxybutyryl-CoATransferase in Oilseed Crops

Methods for the identification of genes encoding enzymes capable offorming 4-hydroxybutyryl-CoA from 4-hydroxybutyric acid (i.e., having4-hydroxybutyryl-CoA transferase activity) which can be expressed in atransgenic plant comprising a PHA synthase transgene were developed bystandard procedures. In certain cases, it may also be useful to expressother PHA biosynthetic genes such as a β-ketothiolase and/oracetoacetyl-CoA reductase in the plant crop of interest. Methods forexpressing a PHA synthase transgene in an oilseed crop have beendescribed (U.S. Pat. No. 5,245,023 and U.S. Pat. No. 5,250,430; U.S.Pat. No. 5,502,273; U.S. Pat. No. 5,534,432; U.S. Pat. No. 5,602,321;U.S. Pat. No. 5,610,041; U.S. Pat. No. 5,650,555: U.S. Pat. No.5,663,063; WO, 9100917, WO 9219747, WO 9302187, WO 9302194 and WO9412014, Poirier et. al., 1992 Science 256; 520–523, Williams andPeoples, 1996 Chemtech 26, 38–44) all of which are incorporated hereinby reference. In order to achieve this goal, it is necessary to transfera gene, or genes in the case of a PHA synthase with more than onesubunit, encoding a PHA synthase from a microorganism into plant cellsand obtain the appropriate level of production of the PHA synthaseenzyme. In addition it may be necessary to provide additional PHAbiosynthetic genes, eg. an acetoacetyl-CoA reductase gene, a4-hydroxybutyryl-CoA transferase gene or other genes encoding enzymesrequired to synthesize the substrates for the PHA synthase enzymes. Inmany cases, it is desirable to control the expression in different planttissues or organelles using methods known to those skilled in the art(Gasser and Fraley, 1989, Science 244; 1293–1299; Gene Transfer toPlants (1995), Potrykus, I. and Spangenberg, G. eds. Springer-VerlagBerlin Heidelberg New York. and “Transgenic Plants: A Production Systemfor Industrial and Pharmaceutical Proteins” (1996), Owen, M. R. L. andPen, J. eds. John Wiley & Sons Ltd. England) all of which areincorporated herein by reference. U.S. Pat. No. 5,610,041 describesplastid expression by adding a leader peptide to direct the proteinexpressed from the nuclear gene to the plastid. More recent technologyenables the direct insertion of foreign genes directly into the plastidchromosome by recombination (Svab et. al., 1990, Proc. Natl;. Acad. Sci.USA. 87: 8526–8530; McBride et. al., 1994, Proc. Natl. Acad Sci. USA.91: 7301–7305). The prokaryotic nature of the plastid RNA and proteinsynthesis machinery also allows for the expression of microbial operonssuch as for example the phbCAB operon of A. eutrophus. This technologyallows for the direct incorporation of a series of genes encoding amulti-enzyme pathway into the plastid genome. It is also important totake into account the importance of 5′-untranslated regions of plastidgenes for mRNA stability and translation (Hauser et. al., 1996. J. Biol.Chem. 271: 1486–1497). In some cases it may be useful to re-engineer the5′-untranslated regions, remove secondary structure elements, or addelements from highly expressed plastid genes to maximize expression oftransgenes encoded by an operon.

1. A recombinant host having stably incorporated into the genome a geneencoding a heterologous 4-hydroxybutyryl-CoA transferase, wherein thehost is selected from the group consisting of a plant, plant cell, andplant component.
 2. The host of claim 1 having stably incorporated intoits genome both a gene encoding a polyhydroxyalkanoate synthase and agene encoding a 4-hydroxybutyryl-CoA transferase.
 3. The host of claim 1further comprising genes expressing enzymes selected from the groupconsisting of beta-ketothiolase, acetoacetyl CoA reductase,polyhydroxyalkanoate synthase, α-ketoglutarate transaminase,glutamate-succinic semialdehyde transaminase, glutamate dehydrogenase,glutamate decarboxylase, and 4-hydroxybutyrate dehydrogenase.
 4. Amethod for enhancing production of polymers containing 4-hydroxybutyratein a host comprising stably incorporating into the genome of the host agene encoding a 4-hydroxybutyryl-CoA transferase, wherein the host isselected from the group consisting of a plant, plant cell, and plantcomponent.
 5. The method of claim 4 wherein the host has stablyincorporated into its genome both a gene encoding a polyhydroxyalkanoatesynthase and a gene encoding a 4-hydroxybutyryl-CoA transferase.
 6. Themethod of claim 4 further comprising enhancing expression of theheterologous enzyme.
 7. The method of claim 4 wherein the host expressesenzymes selected from the group consisting of α-ketoglutaratetransaminase, glutamate-succinic semialdehyde transaminase, glutamatedehydrogenase, glutamate decarboxylase, 4-hydroxybutyrate dehydrogenaseand 4-hydroxybutyryl CoA transferase.
 8. The method of claim 4 whereinthe host expresses enzymes that degrade arginine, glutamine or prolineto produce gamma amino butyric acid.
 9. A 4-hydroxybutyrate polymerproduced by a recombinant host having stably incorporated into thegenome a gene encoding a heterologous enzyme selected from the groupconsisting of a polyhydroxyalkanoate synthase and a 4-hydroxybutyryl-CoAtransferase, wherein the host is selected from the group consisting of aplant, plant cell, and plant component.
 10. A vector comprising anisolated gene encoding a 4-hydroxybutyryl-CoA transferase under thecontrol of a promoter for enhancing expression of the gene encoding the4-hydroxybutyryl-CoA transferase after integration of the promoter andgene encoding the 4-hydroxybutyryl-CoA transferase into the genome of aheterologous host, wherein the host is selected from the groupconsisting of a plant, plant cell, and plant component.
 11. Therecombinant host according to claim 1 wherein the plant cell or plantcomponent is obtained from said plant, and wherein said plant isselected from the group consisting of brassica, sunflower, soybean,corn, safflower, flax, palm, coconut, potato, tapioca and cassava. 12.The method according to claim 4 wherein the plant cell or plantcomponent is obtained from said plant, and wherein said plant isselected from the group consisting of brassica, sunflower, soybean,corn, safflower, flax, palm, coconut, potato, tapioca and cassava. 13.The 4-hydroxybutyrate polymer according to claim 9 wherein the plantcell or plant component is obtained from said plant, and wherein saidplant is selected from the group consisting of brassica, sunflower,soybean, corn, safflower, flax, palm, coconut, potato, tapioca andcassava.
 14. The vector according to claim 10 wherein the plant cell orplant component is obtained from said plant, and wherein said plant isselected from the group consisting of brassica, sunflower, soybean,corn, safflower, flax, palm, coconut, potato, tapioca and cassava.
 15. Arecombinant expression system comprising a host having stablyincorporated into the genome, a gene encoding a heterologous enzymeselected from the group consisting of a polyhydroxyalkanoate synthaseand a 4-hydroxybutyryl-CoA transferase, and a feedstock comprising asubstrate for the polyhydroxyalkanoate synthase and 4-hydroxybutyrl-CoAtransferase selected from the group consisting of carbohydrates,succinate, 4-hydroxybutyrate, α-ketoglutarate, and amino acids, whereinthe enzyme expression and substrate are in a sufficient amount toproduce polyhydroxybutyrate-co-poly-4-hydroxybutyrate orpoly-4-hydroxybutyrate.
 16. A method for enhancing production ofpolymers containing 4-hydroxybutyrate in a host comprising stablyincorporating into the genome of the host, a gene encoding aheterologous enzyme selected from the group consisting of apolyhydroxyalkanoate synthase and a 4-hydroxybutyryl-CoA transferase;providing the host with a feedstock comprising a substrate for thepolyhydroxyalkanoate synthase and 4-hydroxybutyrl-CoA transferaseselected from the group consisting of carbohydrates, succinate,4-hydroxybutyrate, α-ketoglutarate, and amino acids, wherein the enzymeexpression and substrate are in a sufficient amount to producepolyhydroxybutyrate-co-poly-4-hydroxybutyrate or poly-4-hydroxybutyrate.